Methods for producing high levels of carboxylic acids by lignocellulosic biomass processing

ABSTRACT

The technology relates, in certain aspects, to the use of novel extreme thermophile microorganisms, which are able to convert lignocellulosic biomass to carboxylic acids, in particular to lactic acid and/or acetic acid, salts or esters thereof.

FIELD OF THE DISCLOSURE

The present disclosure relates to novel methods for convertinglignocellulosic biomass material for the production of carboxylic acidslike lactic acid.

BACKGROUND

Many carboxylic acids are produced industrially on a large scale. Theyare also pervasive in nature. Carboxylic acids are used in theproduction of polymers, pharmaceuticals, solvents, and food additives.Industrially important carboxylic acids include acetic acid (componentof vinegar, precursor to solvents and coatings), acrylic and methacrylicacids (precursors to polymers, adhesives), adipic acid (polymers),citric acid (beverages), ethylenediaminetetraacetic acid (chelatingagent), fatty acids (coatings), maleic acid (polymers), propionic acid(food preservative), terephthalic acid (polymers).

Lactic acid is widely used in food, pharmaceutical and textileindustries. It is also used as a source of lactic acid polymers whichare being used as biodegradable plastics. The physical properties andstability of polylactides can be controlled by adjusting the proportionsof the L(+)- and D(−)-lactides. Optically pure lactic acid is currentlyproduced by the fermentation of glucose derived from cornstarch usingvarious lactic acid bacteria 7.

However, the fastidious lactic acid bacteria have complex nutritionalrequirements and the use of corn as the feedstock competes directly withthe food and feed uses. Lignocellulosic biomass represents a potentiallyinexpensive and renewable source of sugars for fermentation. Thereofre,the industry of producing fermentation products such as lactic acid isfacing the challenge of redirecting the production process fromfermentation of relatively easily convertible but expensive starchymaterials, to the complex but inexpensive lignocellulosic biomass suchas plant biomass.

Unlike starch, which contains homogenous and easily hydrolysed polymers,lignocellulosic biomass contains variable amounts of cellulose,hemicellulose, lignin and small amounts of protein, pectin, wax andother organic compounds. Lignocellulosic biomass should be understood inits broadest sense, so that it apart from wood, agricultural residues,energy crops also comprises different types of waste from both industryand households. Cellulosic biomass is a vast poorly exploited resource,and in some cases a waste problem. However, hexoses from cellulose canbe converted by yeast to fuel ethanol for which there is a growingdemand. Pentoses from hemicellulose cannot yet be converted to ethanolcommercially but several promising ethanologenic microorganisms with thecapacity to convert pentoses and hexoses are under development.

Typically, the first step in utilization of lignocellulosic biomass is apre-treatment step, in order to fractionate the components oflignocellulosic material and increase their surface area.

The pre-treatment method most often used is steam pretreatment, aprocess comprising heating of the lignocellulosic material by steaminjection to a temperature of 130-230° C. Prior to or during steampretreatment, a catalyst like mineral or organic acid or a caustic agentfacilitating disintegration of the biomass structure can be addedoptionally.

Another type of lignocellulose hydrolysis is acid hydrolysis, where thelignocellulosic material is subjected to an acid such as sulphuric acidwhereby the sugar polymers cellulose and hemicellulose are partly orcompletely hydrolysed to their constituent sugar monomers and thestructure of the biomass is destroyed facilitating access of hydrolyticenzymes in subsequent processing steps.

A further method is wet oxidation wherein the material is treated withoxygen at 150-185° C. Either pretreatment can be followed by enzymatichydrolysis to complete the release of sugar monomers. This pre-treatmentstep results in the hydrolysis of cellulose into glucose whilehemicellulose is transformed into the pentoses xylose and arabinose andthe hexoses glucose, mannose and galactose. Thus, in contrast to starch,the hydrolysis of lignocellulosic biomass results in the release ofpentose sugars in addition to hexose sugars. This implies that usefulfermenting organisms need to be able to convert both hexose and pentosesugars to desired fermentation products such as carboxylic acids.

After the pre-treatment the lignocellulosic biomass processing schemesinvolving enzymatic or microbial hydrolysis commonly involve fourbiologically mediated transformations: (1) the production ofsaccharolytic enzymes (cellulases and hemicellulases); (2) thehydrolysis of carbohydrate components present in pretreated biomass tosugars; (3) the fermentation of hexose sugars (e.g. glucose, mannose,and galactose); and (4) the fermentation of pentose sugars (e.g., xyloseand arabinose).

Each processing step can make the overall process more costly and,therefore, decrease the economic feasibility of producing biofuel orcarbon-based chemicals from cellulosic biological material. Thus, thereis a need to develop methods that reduce the number of processing stepsneeded to convert cellulosic biological material to biofuel and othercommercially desirable materials.

The four biologically mediated transformations may occur in a singlestep in a process configuration called consolidated bioprocessing (CBP),which is distinguished from other less highly integrated configurationsin that CBP does not involve a dedicated process step for cellulaseand/or hemicellulase production. CBP offers the potential for higherefficiency than a processes requiring dedicated cellulase production ina distinct unit operation.

Therefore, the availability of novel microorganisms for convertinglignocellulosic biomass material to carbon-based chemicals like lacticacid would be highly advantageous.

SUMMARY OF THE DISCLOSURE

The present invention relates to methods using novel extremethermophilic microorganisms, and compositions for processinglignocellulosic biomass to carboxylic acids like lactic acid and/oracetic acid.

In a first aspect, embodiments of the disclosure provide the use ofnovel isolated cellulolytic thermophilic bacterial cells belonging tothe genus Caldicellulosiruptor for producing high levels of acids, inparticular of lactic acid from lignocellulosic biomass material.

Embodiments of this disclosure relate to the use of an isolatedCaldicellulosiruptor sp. cell comprising a 16S rDNA with a sequenceselected form the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ IDNO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO 7, orhomolgues thereof.

In one aspect, embodiments of this disclosure relate to the use of anisolated Caldicellulosiruptor sp. DIB004C, Caldicellulosiruptor sp.DIB041C, Caldicellulosiruptor sp. DIB087C, Caldicellulosiruptor sp.DIB101C, Caldicellulosiruptor sp. DIB103C, Caldicellulosiruptor sp.DIB104C or Caldicellulosiruptor sp. DIB107C, each respectivelycharacterized by having a 16S rDNA sequence at least 99 to 100%,preferably 99.5 to 99.99 percent identical to SEQ ID NO. 1, SEQ ID NO.2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO 7as outlined in table 1.

In still another aspect the present invention relates to the use of anisolated strain comprising a Caldicellulosiruptor sp. cell according toany of the preceding aspects.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB004C deposited as DSM 25177, amicroorganism derived therefrom or a Caldicellulosiruptor sp. DIB004Chomolog or mutant.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB041C deposited as DSM 25771, amicroorganism derived therefrom or a Caldicellulosiruptor sp. DIB041Chomolog or mutant.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB087C deposited as DSM 25772, amicroorganism derived there from or a Caldicellulosiruptor sp. DIB087Chomolog or mutant.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB101C deposited as DSM 25178, amicroorganism derived there from or a Caldicellulosiruptor sp. DIB101Chomolog or mutant.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB103C deposited as DSM 25773, amicroorganism derived there from or a Caldicellulosiruptor sp. DIB103Chomolog or mutant.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB104C deposited as DSM 25774, amicroorganism derived there from or a Caldicellulosiruptor sp. DIB104Chomolog or mutant.

In a further aspect, embodiments of this disclosure relate to the use ofthe strain Caldicellulosiruptor sp. DIB107C deposited as DSM 25775, amicroorganism derived there from or a Caldicellulosiruptor sp. DIB107Chomolog or mutant.

In another aspect the present disclosure relates to methods of producinga carboxylic acid comprising culturing a cell according to thedisclosure or a strain according to the disclosure under suitableconditions.

In still another aspect, embodiments of this disclosure relate tomethods for converting lignocellulosic biomass material to a carboxylicacid, in particular lactic acid and/or acetic acid comprising the stepof contacting the lignocellulosic biomass material with a microbialculture for a period of time at an initial temperature and an initialpH, thereby producing an amount of a carboxylic acid, in particular tolactic acid, wherein the microbial culture comprises an extremelythermophilic microorganism of the genus Caldicellulosiruptor, inparticular all microorganisms of the strain Caldicellulosiruptor sp. aslisted in table 1 with their respective deposition numbers,microorganisms derived from either of these strains or mutants orhomologues thereof.

In still another aspect, embodiments of this disclosure relate tomethods of making carboxylic acid from biomass material, wherein themethod comprises combining a microbial culture and the biomass in amedium; and fermenting the biomass under conditions and for a timesufficient to produce carboxylic acids, in a single step process as partof a consolidated bioprocessing (CBP) system, with a cell, strain,microbial culture and/or a microorganism according to the presentdisclosure under suitable conditions.

In still another aspect, embodiments of this disclosure relate tomethods of making carboxylic acid from biomass material, wherein themethod comprises combining a microbial culture and the biomass in amedium; and fermenting the biomass under conditions and for a timesufficient to produce carboxylic acids, preferably lactic acids, saltsor esters thereof, in a single step process as part of a consolidatedbioprocessing (CBP) system, with a cell, strain, microbial cultureand/or a microorganism according to the present disclosure undersuitable conditions.

In still another aspect, embodiments of this disclosure relate tomethods of making carboxylic acids from biomass material, wherein themethod comprises combining a microbial culture and the biomass in amedium; and fermenting the biomass under conditions and for a timesufficient to produce a carboxylic acid in a single step process as partof a consolidated bioprocessing (CBP) system, with a cell, strain,microbial culture and/or a microorganism according to the presentdisclosure under suitable conditions in combination with application ofmethod suitable to in-situ remove both or either fermentation productfrom the fermentation broth. Suitable methods include but are notlimited to distillation, mediated distillation, extraction andprecipitation.

Further, embodiments of this disclosure relate to the use ofcompositions for converting lignocellulosic biomass or a microbialculture to a carboxylic acid, in particular to lactic acid, comprising acell, strain or microorganism according to the present disclosure.

Before the disclosure is described in detail, it is to be understoodthat this disclosure is not limited to the particular component parts ofthe devices described or process steps of the methods described as suchdevices and methods may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting. It must be notedthat, as used in the specification and the appended claims, the singularforms “a,” “an” and “the” include singular and/or plural referentsunless the context clearly dictates otherwise. It is moreover to beunderstood that, in case parameter ranges are given which are delimitedby numeric values, the ranges are deemed to include these limitationvalues.

To provide a comprehensive disclosure without unduly lengthening thespecification, the applicant hereby incorporates by reference each ofthe patents and patent applications cited herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a phylogenetic tree based on 16S rDNA genes for allCaldicellulosiruptor sp. strains comprised in the invention as listed intable 1

FIG. 2 shows a 16S rDNA from Caldicellulosiruptor sp. DIB004C cell.

FIG. 3 shows a 16S rDNA from Caldicellulosiruptor sp. DIB041C cell.

FIG. 4 shows a 16S rDNA from Caldicellulosiruptor sp. DIB087C cell.

FIG. 5 shows a 16S rDNA from Caldicellulosiruptor sp. DIB101C cell.

FIG. 6 shows a 16S rDNA from Caldicellulosiruptor sp. DIB103C cell.

FIG. 7 shows a 16S rDNA from Caldicellulosiruptor sp. DIB104C cell.

FIG. 8 shows a 16S rDNA from Caldicellulosiruptor sp. DIB107C cell.

FIG. 9 shows a graph indicating production of ethanol, lactic and aceticacid by DIB004C during growth on steam-pretreated miscanthus grass.

FIG. 10 shows a table indicating performance data from all strainslisted in table 1 and reference strain C. saccharolyticus DSM8903 duringcultivation on cellulose, cellobiose, glucose, xylan, xylose andpretreated lignocellulosic biomass.

FIG. 11 shows a table indicating performance data from strains DIB004Cand DIB101C on various types of pretreated lignocellulosic biomass

DETAILED DESCRIPTION OF THIS DISCLOSURE

The present disclosure relates to methods using microorganisms, andcompositions for processing lignocellulosic biomass. The disclosurerelates, in certain aspects, to the use of microorganisms, which areable to convert lignocellulosic biomass such as, for example, miscanthusgrass, to soluble products that can be used by the same or by anothermicroorganism to produce an economically desirable product such ascarboxylic acids, in particular to lactic acid and/or acetic acid, saltsor esters thereof.

The application of this technology has the potential to renderproduction of carboxylic acids more economically feasible and to allow abroader range of microorganisms to utilize recalcitrant biomass. The useof cellulosic materials as sources of bioenergy is currently limited bytypically requiring preprocessing of the cellulosic material. Suchpreprocessing methods can be expensive. Thus, methods that reducedependence on preprocessing of cellulosic materials may have a dramaticimpact on the economics of the use of recalcitrant biomass for biofuelsproduction. One challenge in converting biomass into fermentationproducts is the recalcitrance and heterogeneity of the biologicalmaterial.

The present inventors have found microorganisms of the genusCaldicellulosiruptor, which have a variety of advantageous propertiesfor their use in the conversion of lignocellulosic biomass material tocarboxylic acids, preferably in a single step process as part of aconsolidated bioprocessing (CBP) system.

Carboxylic acids are organic acids characterized by the presence of atleast one carboxyl group. The general formula of a carboxylic acid isR—COOH, where R is some monovalent functional group. A carboxyl group(or carboxy) is a functional group consisting of a carbonyl (RR′C═O) anda hydroxyl (R—O—H), which has the formula —C(═O)OH, usually written as—COOH or —CO2H. The term carboxylic acids includes salts and esters ofthe acids. Lactic acid is a carboxylic acid with the chemical formulaC3H6O3. It has a hydroxyl group adjacent to the carboxyl group, makingit an alpha hydroxy acid (AHA).

In particular, these microorganisms are extremely thermophilic and showa broad substrate specificities and high natural production ofcarboxylic acids. Moreover, carboxylic acids fermentation at hightemperatures, for example over 70° C. has many advantages overmesophilic fermentation. One advantage of thermophilic fermentation isthe minimization of the problem of contamination in batch cultures,fed-batch cultures or continuous cultures, since only a fewmicroorganisms are able to grow at such high temperatures inun-detoxified lignocellulose biomass material.

It is also an advantage that the cells, strains and microorganismsaccording to the present disclosure grow on pre-treated as well as onuntreated lignocellulosic biomass material.

The isolated cells, strains, microorganisms, compositions and microbialcultures are capable of growing and producing fermentation products onvery high dry-matter concentrations of lignocellulosic biomass material.

In the present context the term “lignocellulosic biomass material” isintended to designate a untreated lignocellulosic biomass and/or alignocellulosic biomass which has been subjected to a pretreatment stepwhereby lignocellulosic material has been at least partially separatedinto cellulose, hemicellulose and lignin thereby having increased thesurface area and/or accessibility of the material. The lignocellulosicmaterial may typically be derived from plant material, such as straw,hay, perennial grass, garden refuse, comminuted wood, fruit hulls andseed hulls.

The pretreatment method most often used is steam pretreatment, a processcomprising heating of the lignocellulosic material by steam injection toa temperature of 130-230 degrees centigrade with or without subsequentsudden release of pressure. Prior to or during steam pretreatment, acatalyst like a mineral or organic acid or a caustic agent facilitatingdisintegration of the biomass structure can be added optionally.Catalysts often used for such a pretreatment include but are not limitedto sulphuric acid, sulphurous acid, hydrochloric acid, acetic acid,lactic acid, sodium hydroxide (caustic soda), potassium hydroxide,calcium hydroxide (lime), ammonia or the respective salts or anhydridesof any of these agents.

Such steam pretreatment step may or may not be preceded by anothertreatment step including cooking of the biomass in water or steaming ofthe biomass at temperatures of 100-200° C. with or without the additionof a suitable catalyst like a mineral or organic acid or a caustic agentfacilitating disintegration of the biomass structure. In between thecooking step and the subsequent steam pretreatment step one or moreliquid-solid-separation and washing steps can be introduced to removesolubilized biomass components in order to reduce or prevent formationof inhibitors during the subsequent steam pretreatment step. Inhibitorsformed during heat or steam pretreatment include but are not limited tofurfural formed from monomeric pentose sugars, hydroxymethylfurfuralformed from monomeric hexose sugars, acetic acid, levulinic acid,phenols and phenol derivatives.

Another type of lignocellulose hydrolysis is acid hydrolysis, where thelignocellulosic material is subjected to an acid such as sulfuric acidor sulfurous acid whereby the sugar polymers cellulose and hemicelluloseare partly or completely hydrolysed to their constituent sugar monomers.A third method is wet oxidation wherein the material is treated withoxygen at 150-185 degrees centigrade. The pretreatments can be followedby enzymatic hydrolysis to complete the release of sugar monomers. Thispre-treatment step results in the hydrolysis of cellulose into glucosewhile hemicellulose is transformed into the pentoses xylose andarabinose and the hexoses glucose, mannose and galactose. Thepretreatment step may in certain embodiments be supplemented withtreatment resulting in further hydrolysis of the cellulose andhemicellulose. The purpose of such an additional hydrolysis treatment isto hydrolyze oligosaccharide and possibly polysaccharide speciesproduced during the acid hydrolysis, wet oxidation, or steampretreatment of cellulose and/or hemicellulose origin to formfermentable sugars (e.g. glucose, xylose and possibly othermonosaccharides). Such further treatments may be either chemical orenzymatic. Chemical hydrolysis is typically achieved by treatment withan acid, such as treatment with aqueous sulphuric acid or hydrochloricacid, at a temperature in the range of about 100-150 degrees centigrade.Enzymatic hydrolysis is typically performed by treatment with one ormore appropriate carbohydrase enzymes such as cellulases, glucosidasesand hemicellulases including xylanases.

It has been found that the microorganisms according to the presentdisclosure can grow efficiently on various types of pretreated anduntreated biomass (e.g. wood incl. poplar, spruce and cotton wood;various types of grasses and grass residues incl. miscanthus, wheatstraw, sugarcane bagasse, corn stalks, corn cobs, whole corn plants,sweet sorghum).

As used herein “efficient” growth refers to growth in which cells may becultivated to a specified density within a specified time.

The microorganisms according to the present disclosure can growefficiently on crystalline cellulose and steam pretreated perennialgrasses and grow efficiently on xylan. The main products when grown onuntreated biomass substrates were lactate, for example, when themicroorganisms grown on cellobiose and or xylane the lactate yield ishigh.

Cellobiose is a disaccharide derived from the condensation of twoglucose molecules linked in a β(1→4) bond. It can be hydrolyzed to giveglucose. Cellobiose has eight free alcohol (OH) groups, one etherlinkage and two hemiacetal linkages, which give rise to strong inter-and intra-molecular hydrogen bonds. It is a type of dietary carbohydratealso found in mushrooms.

Xylan is a generic term used to describe a wide variety of highlycomplex polysaccharides that are found in plant cell walls and somealgae. Xylans are polysaccharides made from units of xylose.

The microorganisms according to the present disclosure also can growefficiently on spent biomass—insoluble material that remains after aculture has grown to late stationary phase (e.g., greater than 10⁸cells/mL) on untreated biomass.

The microorganisms according to the present disclosure also grewefficiently on cellobiose, untreated switchgrass, and untreated poplarand poplar that had been heated at 98 degrees centigrade for twominutes.

Furthermore, the microorganisms according to the present disclosure grewefficiently on both the soluble and insoluble materials obtained afterheat treating the biomass.

It was surprisingly found that the bacterial subspecies according to thepresent disclosure is capable of growing in a medium comprising alignocellulosic biomass material having a dry-matter content of at least10 percent wt/wt, such as at least 15 percent wt/wt, including at least20 percent wt/wt, and even as high as at least 25 percent wt/wt.

The microorganisms according to the invention are anaerobic thermophilebacteria, and they are capable of growing at high temperatures even ator above 70° C. The fact that the strains are capable of operating atthis high temperature is of high importance in the conversion of thelignocellulosic material into fermentation products. The conversion rateof carbohydrates into carboxylic acids is much faster when conducted athigh temperatures. For example, the volumetric lactic acid productivityof a thermophilic Bacillus is up to ten-fold higher than a conventionalyeast fermentation process which operates at 30 degrees centigradeConsequently, a smaller production plant is required for a given plantcapacity, thereby reducing plant construction costs. As also mentionedpreviously, the high temperature reduces the risk of contamination fromother microorganisms, resulting in less downtime, increased plantproductivity and a lower energy requirement for feedstock sterilization.The high operation temperature may also facilitate the subsequentrecovery of the resulting fermentation products.

Lignocellulosic biomass material and lignocellulose hydrolysates containinhibitors such as furfural, phenols and carboxylic acids, which canpotentially inhibit the fermenting organism. Therefore, it is anadvantage of the microorganisms according to the present disclosure thatthey are tolerant to these inhibitors.

The microorganisms according to the present disclosure are novel speciesof the genus Caldicellulosiruptor or novel subspecies ofCaldicellulosiruptor saccharolyticus.

For example, the genus Caldicellulosiruptor includes different speciesof extremely thermophilic (growth at temperature significantly above 70°C.) cellulolytic and hemicellulolytic strictly anaerobic nonsporeformingbacteria. The first bacterium of this genus, Caldicellulosiruptorsaccharolyticus strain Tp8T (DSM 8903) has a temperature optimum of 70°C. and was isolated from a thermal spring in New Zealand (Rainey et al.1994; Sissons et al. 1987). It hydrolyses a variety of polymericcarbohydrates with the production of acetate, lactate and trace amountsof ethanol (Donnison et al. 1988). Phylogenetic analysis showed that itconstitutes a novel lineage within the Bacillus/Clostridium subphylum ofthe Gram-positive bacteria (Rainey et al. 1994).

According to the present disclosure, the microorganisms producecarboxylic acids like lactic acid and show several features thatdistinguish them from currently used microorganisms: (i) high yield andlow product inhibition, (ii) simultaneous utilization of lignocellolyticbiomass material and/or sugars, and (iii) growth at elevatedtemperatures. The microorganisms according to the present disclosure arerobust thermophile organisms with a decreased risk of contamination.They efficiently convert an extraordinarily wide range of biomasscomponents to carboxylic acids like lactic acid and/or acetic acid.

Each independently an embodiment of the invention is the use of anisolated cell which is Caldicellulosiruptor sp. DIB004C (DSMZ Accessionnumber 25177), an isolated cell which is Caldicellulosiruptor sp. DIB041C (DSMZ Accession number 25771), an isolated cell which isCaldicellulosiruptor sp. DIB087C (DSMZ Accession number 25772), anisolated cell which is Caldicellulosiruptor sp. DIB101C (DSMZ Accessionnumber 25178), an isolated cell which is Caldicellulosiruptor sp.DIB103C (DSMZ Accession number 25773), an isolated cell which isCaldicellulosiruptor sp. DIB104C (DSMZ Accession number 25774) or anisolated cell which is Caldicellulosiruptor sp. DIB107C (DSMZ Accessionnumber 25775), cells derived from either, mutants or a homolog ofeither.

As used herein “mutant” or “homolog” means a microorganism derived fromthe cells or strains according to the present disclosure, which arealtered due to a mutation. A mutation is a change produced in cellularDNA, which can be spontaneous, caused by an environmental factor orerrors in DNA replication, or induced by physical or chemicalconditions. The processes of mutation included in this and indentedsubclasses are processes directed to production of essentially randomchanges to the DNA of the microorganism including incorporation ofexogenous DNA. All mutants of the microorganisms comprise the advantagesof being extereme thermophile (growing and fermenting at temperaturesabove 70° C.) and are capable of fermenting lignocellulosic biomass atleast to a carboxylic acid like lactic acid In an advantageousembodiment, mutants of the microorganisms according to the presentdisclosure have in a DNA-DNA hybridization assay, a DNA-DNA relatednessof at least 80%, preferably at least 90%, at least 95%, more preferredat least 98%, most preferred at least 99%, and most preferred at least99.9% with one of the isolated bacterial strains Caldicellulosiruptorsp. DIB004C, DIB041C, DIB087C, DIB101C, DIB103C, DIB104C and DIB107C.

As mentioned above, in one aspect, the present disclosure relates to theuse of an isolated cell comprising a 16S rDNA sequence selected from thegroup consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4,SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, and a combination of anythereof.

In one aspect, mutants of the microorganisms according to the presentdisclosure pertains to an isolated Caldicellulosiruptor sp. cell havinga 16S rDNA sequence at least 99, at least 99.3, at least 99.5, at least,99.7, at least 99.9, at least 99.99 percent identical to one of thesequences listed in table 1.

The invention is based on the use of isolated bacterial strainsCaldicellulosiruptor sp. DIB004C, DIB041C, DIB087C, DIB101C, DIB103C,DIB104C and DIB107C that contain 16S rDNA sequences at least 99 to 100%,preferably 99.5 to 99.99, more preferably at least 99.99 percentidentical to the respective sequences listed in table 1, in the methodsaccording to the present disclosure.

TABLE 1 DSMZ accession 16SrDNA Genus Species Name number Deposition dateSEQ ID NO. Caldicellulosiruptor sp. DIB004C DSM 25177 Sep. 15, 2011 1Caldicellulosiruptor sp. DIB041C DSM 25771 Mar. 15, 2012 2Caldicellulosiruptor sp. DIB087C DSM 25772 Mar. 15, 2012 3Caldicellulosiruptor sp. DIB101C DSM 25178 Sep. 15, 2011 4Caldicellulosiruptor sp. DIB103C DSM 25773 Mar. 15, 2012 5Caldicellulosiruptor sp. DIB104C DSM 25774 Mar. 15, 2012 6Caldicellulosiruptor sp. DIB107C DSM 25775 Mar. 15, 2012 7

The strains listed in table 1 have been deposited in accordance with theterms of the Budapest Treaty on Sep. 15, 2011 with DSMZ—DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B,38124 Braunschweig, Germany, under the respectively indicated DSMZaccession numbers and deposition dates, respectively, by DIREVOIndustrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne (DE).

The microorganisms of the species Caldicellulosiruptor sp. according tothe present disclosure in particular refer to a microorganism whichbelongs to the genus Caldicellulosiruptor and which preferably has oneor more of the following characteristics:

-   -   a) it is a microorganism of the genus Caldicellulosiruptor;    -   b) in a DNA-DNA hybridization assay, it shows a DNA-DNA        relatedness of at least 70%, preferably at least 90%, at least        95%, more preferred at least 98%, most preferred at least 99%        with either Caldicellulosiruptor sp. strain listed in table 1        with their respective accession numbers; and/or    -   c) it displays a level of 16S rDNA gene sequence similarity of        at least 98%, preferably at least 99% or at least 99,5%, more        preferably 100% with either either Caldicellulosiruptor sp.        strain listed in table 1 with their respective accession        numbers; and/or    -   d) it is capable of surviving in high temperature conditions        above 75° C.    -   e) it is capable of surviving in high temperature conditions        above 70° C., and or    -   f) it is a Gram-positive bacterium.

Preferably, at least two or at least three, and more preferred all ofthe above defined criteria a) to f) are fulfilled.

In an advantageous embodiment, the microorganisms according to thepresent disclosure in particular refer to a microorganism which belongsto the genus Caldicellulosiruptor and which preferably has one or moreof the following characteristics:

-   -   a) It is a microorganism of the genus Caldicellulosiruptor    -   b) it is a microorganism of the species Caldicellulosiruptor        saccharolyticus;    -   c) in a DNA-DNA hybridization assay, it shows a DNA-DNA        relatedness of at least 80%, preferably at least 90%, at least        95%, more preferred at least 98%, most preferred at least 99%,        and most preferred at least 99,9% with one of the strains of        table 1; and/or    -   d) it displays a level of 16S rDNA gene sequence similarity of        at least 98%, preferably at least 99%, at least 99.5% or at        least 99.7%, more preferably 99.99% with one of the strains        listed in table 1; and/or    -   e) it is capable of surviving and/or growing and/or producing a        carboxylic acid at temperature conditions above 70° C., in        particular of above 72° C.

Preferably, at least two or at least three, and more preferred all ofthe above defined criteria a) to e) are fulfilled.

The term “DNA-DNA relatedness” in particularly refers to the percentagesimilarity of the genomic or entire DNA of two microorganisms asmeasured by the DNA-DNA hybridization/renaturation assay according to DeLey et al. (1970) Eur. J. Biochem. 12, 133-142 or Huβ et al. (1983)Syst. Appl. Microbiol. 4, 184-192. In particular, the DNA-DNAhybridization assay preferably is performed by the DSMZ (DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig,Germany) Identification Service.

The term “16S rDNA gene sequence similarity” in particular refers to thepercentage of identical nucleotides between a region of the nucleic acidsequence of the 16S ribosomal RNA (rDNA) gene of a first microorganismand the corresponding region of the nucleic acid sequence of the 16SrDNA gene of a second microorganism. Preferably, the region comprises atleast 100 consecutive nucleotides, more preferably at least 200consecutive nucleotides, at least 300 consecutive nucleotides or atleast 400 consecutive nucleotides, most preferably about 480 consecutivenucleotides.

The strains according to disclosure have the potential to be capable ofproducing carboxylic acids like lactic acid and acetic acid.

The use of the Caldicellulosiruptor sp. strains according to the presentdisclosure have several highly advantageous characteristics needed forthe conversion of lignocellulosic biomass material. Thus, these basestrains possess all the genetic machinery for the hydrolysis ofcellulose and hemicelluloses and for the conversion of both pentose andhexose sugars to various fermentation products such as carboxylic acidslike lactic acid. As will be apparent from the below examples, theexamination of the complete 16S rDNA sequence showed that the closelyrelated strains may all be related to Caldicellulosiruptorsaccharolyticus although the 16S rDNA sequences may place them in aseparate subspecies or even a different species

Furthermore, the Caldicellulosiruptor sp. strains according to thepresent disclosure are cellulolytic and xylanolytic.

In a preferred embodiment, the Caldicellulosiruptor sp. microorganismused in a method according to the present disclosure is

a) Caldicellulosiruptor sp. DIB004C, deposited on Sep. 15, 2011 underthe accession number DSM 25177 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB004C or

c) a Caldicellulosiruptorsp. DIB004C mutant.

In another preferred embodiment, the Caldicellulosiruptor sp.microorganism is

a) Caldicellulosiruptor sp. DIB041C, deposited on Mar. 15, 2012 underthe accession number DSM 25771 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB041C or

c) a Caldicellulosiruptor sp. DIB041C mutant.

In another preferred embodiment, the Caldicellulosiruptor sp.microorganism used in a method according to the present disclosure is

a) Caldicellulosiruptor sp. DIB087C, deposited on Mar. 15, 2012 underthe accession number DSM 25772 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB087C or

c) a Caldicellulosiruptor sp. DIB087C mutant.

In another preferred embodiment, the Caldicellulosiruptor sp.microorganism used in a method according to the present disclosure is

a) Caldicellulosiruptor sp. DIB101C, deposited on Sep. 15, 2011 underthe accession number DSM 25178 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB101C or

c) a Caldicellulosiruptor sp. DIB101C mutant.

In another preferred embodiment, the Caldicellulosiruptor sp.microorganism used in a method according to the present disclosure is

a) Caldicellulosiruptor sp. DIB103C, deposited on Mar. 15, 2012 underthe accession number DSM 25773 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB103C or

c) a Caldicellulosiruptor sp. DIB103C mutant.

In another preferred embodiment, the Caldicellulosiruptor sp.microorganism used in a method according to the present disclosure is

a) Caldicellulosiruptor sp. DIB104C, deposited on Mar. 15, 2012 underthe accession number DSM 25774 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB104C or

c) a Caldicellulosiruptor sp. DIB104C mutant.

In another preferred embodiment, the Caldicellulosiruptor sp.microorganism used in a method according to the present disclosure is

a) Caldicellulosiruptor sp. DIB107C, deposited on Mar. 15, 2012 underthe accession number DSM 25775 according to the requirements of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) byDIREVO Industrial Biotechnology GmbH, Nattermannallee 1, 50829 Cologne(DE),

b) a microorganism derived from Caldicellulosiruptor sp. DIB107C or

c) a Caldicellulosiruptor sp. DIB107C mutant.

All strains listed above and in table 1 belong to the genusCaldicellulosiruptor and are strictly anaerobic, non-sporeforming,non-motile, gram-positive bacteria. Cells are straight rods 0.4-0.5 μmby 2.0-4.0 μm, occuring both singly and in pairs. After 7 daysincubation at 72° C. on solid medium with agar and cellulose assubstrate both strains form circular milky colonies of 0.5-1 mm indiameter. Clearing zones around the colonies are produced indicatingcellulose degradation.

The term “a microorganism” as used herein may refer to only oneunicellular organism as well as to numerous single unicellularorganisms. For example, the term “a microorganism of the genusCaldicellulosiruptor” may refer to one single Caldicellulosiruptorbacterial cell of the genus Caldicellulosiruptor as well as to multiplebacterial cells of the genus Caldicellulosiruptor.

The terms “a strain of the genus Caldicellulosiruptor” and “aCaldicellulosiruptor cell” are used synonymously herein. In general, theterm “a microorganism” refers to numerous cells. In particular, saidterm refers to at least 10³ cells, preferably at least 10⁴ cells, atleast 10⁵ or at least 10⁶ cells.

As mentioned above lignocellolytic biomass according to the presentdisclosure can be but is not limited to grass, switch grass, cord grass,rye grass, reed canary grass, mixed prairie grass, miscanthus, Napiergrass, sugar-methoding residues, sugarcane bagasse, sugarcane straw,agricultural wastes, rice straw, rice hulls, barley straw, corn cobs,cereal straw, wheat straw, canola straw, oat straw, oat hulls, cornfiber, stover, soybean stover, corn stover, forestry wastes, recycledwood pulp fiber, paper sludge, sawdust, hardwood, softwood, pressmudfrom sugar beet, cotton stalk, banana leaves, oil palm residues andlignocellulosic biomass material obtained through processing of foodplants. In advantageous embodiments, the lignocellulosic biomassmaterial is hardwood and/or softwood, preferably poplar wood. Inadvantageous embodiments, the lignocellulosic biomass material is agrass or perennial grass, preferably miscanthus.

In advantageous embodiments, the lignocellulosic biomass material issubjected to mechanical, thermochemical, and/or biochemicalpretreatment. The lignocellulosic biomass material could be exposed tosteam treatment. In further embodiments, the lignocellulosic biomassmaterial is pretreated with mechanical comminution and a subsequenttreatment with lactic acid, acetic acid, sulfuric acid or sulfurous acidor their respective salts or anhydrides under heat and pressure with orwithout a sudden release of pressure. In another embodiment, thelignocellulosic biomass material is pretreated with mechanicalcomminution and a subsequent treatment with either sodium hydroxide,ammonium hydroxide, calcium hydroxide or potassium hydroxide under heatand pressure with or without a sudden release of pressure.

In advantageous embodiments, the lignocellulosic biomass material ispretreated with mechanical comminution and subsequent exposure to amulti-step combined pretreatment process. Such multi-step combinedpretreatment may include a treatment step consisting of cooking in wateror steaming of the lignocellulosic biomass material at a temperature of100-200° C. for a period of time in between 5 and 120 min. Suitablecatalysts including but not limited to lactic acid, acetic acid,sulfuric acid, sulfurous acid, sodium hydroxide, ammonium hydroxide,calcium hydroxide or potassium hydroxide or their respective salts oranhydrides may or may not be added to the process. The process mayfurther include a step comprising a liquid-solid separation operation,e.g. filtration, separation, centrifugation or a combination thereof,separating the process fluid containing partially or fully hydrolyzedand solubilized constituents of the lignocellulosic biomass materialfrom the remaining insoluble parts of the lignocellulosic biomass. Theprocess may further include a step comprising washing of the remaininglignocellulosic biomass material. The solid material separated fromsolubilized biomass constituents may then be treated in a second stepwith steam under heat and pressure with or without a sudden release ofpressure at a temperature of 150-250° C. for a period of time in between1 and 15 min. In order to increase pretreatement effectiveness, asuitable catalyst including but not limited to lactic acid, acetic acid,sulfuric acid, sulfurous acid, sodium hydroxide, ammonium hydroxide,calcium hydroxide or potassium hydroxide or their respective salts oranhydrides may be added also to the second step.

In advantageous embodiments, the lignocellulosic biomass is milledbefore converted into carboxylic acids like lactic acid. In oneembodiment, the lignocellulosic biomass is pretreated biomass fromPopulus sp, preferably pretreated with steam pretreatment or multi-stepcombined pretreatment. In another embodiment, the lignocellulosicbiomass is pretreated biomass from any perennial grass, e.g. Miscanthussp., preferably treated with steam pretreatment or multi-step combinedpretreatment.

In advantageous embodiments the cells, strains, microorganisms may bemodified in order to obtain mutants or derivatives with improvedcharacteristics. Thus, in one embodiment there is provided a bacterialstrain according to the disclosure, wherein one or more genes have beeninserted, deleted or substantially inactivated. The variant or mutant istypically capable of growing in a medium comprising a lignocellulosicbiomass material.

In another embodiment, there is provided a process for preparingvariants or mutants of the microorganisms according to the presentdisclosure, wherein one or more genes are inserted, deleted orsubstantially inactivated as described herein.

In some embodiments one or more additional genes are inserting into thestrains according to the present disclosure. Thus, in order to improvethe yield of the specific fermentation product, it may be beneficial toinsert one or more genes encoding a polysaccharase into the strainaccording to the invention. Hence, in specific embodiments there isprovided a strain and a process according to the invention wherein oneor more genes encoding a polysaccharase which is selected fromcellulases (such as EC 3.2.1.4); beta-glucanases, including glucan-1,3beta-glucosidases (exo-1,3 beta-glucanases, such as EC 3.2.1.58),1,4-beta-cellobiohydrolases (such as EC 3.2.1.91) andendo-I,3(4)-beta-glucanases (such as EC 3.2.1.6); xylanases, includingendo-1,4-beta-xylanases (such as EC 3.2.1.8) and xylan1,4-beta-xylosidases (such as EC 3.2.1.37); pectinases (such as EC3.2.1.15); alpha-glucuronidases, alpha-L-arabinofuranosidases (such asEC 3.2.1.55), acetylesterases (such as EC 3.1.1.-), acetylxylanesterases(such as EC 3.1.1.72), alpha-amylases (such as EC 3.2.1.1),beta-amylases (such as EC 3.2.1.2), glucoamylases (such as EC 3.2.1.3),pullulanases (such as EC 3.2.1.41), beta-glucanases (such as EC3.2.1.73), hemicellulases, arabinosidases, mannanases including mannanendo-I,4-beta-mannosidases (such as EC 3.2.1.78) and mannanendo-I,6-alpha-mannosidases (such as EC 3.2.1.101), pectin hydrolases,polygalacturonases (such as EC 3.2.1.15), exopolygalacturonases (such asEC 3.2.1.67) and pectate lyases (such as EC 4.2.2.10), are inserted.

In accordance with the present disclosure, a method of producing afermentation product comprising culturing a strain according to theinvention under suitable conditions is also provided.

The strains according to the disclosure are strictly anaerobicmicroorganisms, and hence it is preferred that the fermentation productis produced by a fermentation process performed under strictly anaerobicconditions. Additionally, the strain according to invention is anextremely thermophillic microorganism, and therefore the process mayperform optimally, when it is operated at temperature in the range ofabout 40-95° C., such as the range of about 50-90° C., including therange of about ° C. degrees centigrade, such as the range of about65-75° C. In an advantageous embodiment the process is operated at 72°C.

For the production of certain fermentation products, it may be useful toselect a specific fermentation process, such as batch fermentationprocess, including a fed-batch process or a continuous fermentationprocess. Also, it may be useful to select a fermentation reactor such asa stirred vessel reactor, an immobilized cell reactor, a fluidized bedreactor or a membrane bioreactor.

In accordance with the invention, the method is useful for theproduction of a wide range of carboxylic acids such as lactic acid andacetic acid may be produced in accordance with the disclosure.

The expression “comprise”, as used herein, besides its literal meaningalso includes and specifically refers to the expressions “consistessentially of” and “consist of”. Thus, the expression “comprise” refersto embodiments wherein the subject-matter which “comprises” specificallylisted elements does not comprise further elements as well asembodiments wherein the subject-matter which “comprises” specificallylisted elements may and/or indeed does encompass further elements.Likewise, the expression “have” is to be understood as the expression“comprise”, also including and specifically referring to the expressions“consist essentially of” and “consist of”.

The following methods and examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present disclosurein any way.

METHODS AND EXAMPLES

In the following examples, materials and methods of the presentdisclosure are provided including the determination of the properties ofthe microbial strains according to the present disclosure. It should beunderstood that these examples are for illustrative purpose only and arenot to be construed as limiting this disclosure in any manner. Allpublications, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

Example 1 Isolation and Cultivation

All procedures for enrichment and isolation of the strains listed intable 1 employed anaerobic technique for strictly anaerobic bacteria(Hungate 1969). The strains were enriched from environmental samples attemperatures higher than 70° C. with crystalline cellulose and beechwood as substrate. Isolation was performed by picking colonies grown onsolid agar medium at 72° C. in Hungate roll tubes (Hungate 1969).

The cells are cultured under strictly anaerobic conditions applying thefollowing medium:

Basic medium NH4Cl 1.0 g NaCl 0.5 g MgSO4 × 7 H2O 0.3 g CaCl2 × 2 H2O0.05 g NaHCO3 0.5 g K2HPO4 1.5 g KH2PO4 3.0 g Yeast extract (bacto, BD)0.5 g Cellobiose 5.0 g Vitamins (see below) 1.0 ml Trace elements (seebelow) 0.5 ml Resazurin 1.0 mg Na2S × 9 H2O 0.75 g Distilled water1000.0 ml Trace elements stock solution NiCl₂ × 6H₂O 2 g FeSO₄ × 7H₂O 1g NH₄Fe(III) citrate, brown, 21.5% Fe 10 g MnSO₄ × H₂O 5 g CoCl₂ × 6H₂O1 g ZnSO₄ × 7H₂O 1 g CuSO₄ × 5H₂O 0.1 g H₃BO₃ 0.1 g Na₂MoO₄ × 2H₂O 0.1 gNa₂SeO₃ × 5H₂O 0.2 g Na₂WoO₄ × 2H₂O 0.1 g Distilled water 1000.0 ml Add0.5 ml of the trace elements stock solution to 1 liter of the mediumVitamine stock solution nicotinic acid 200 mg cyanocobalamin 25 mgp-aminobenzoic acid 25 mg (4-aminobenzoic acid) calcium D-pantothenate25 mg thiamine-HCl 25 mg riboflavin 25 mg lipoic acid 25 mg folic acid10 mg biotin 10 mg pyridoxin-HCl 10 mg Distilled water 200.0 ml Add 1 mlof the vitamine stock solution to 1 liter of the medium

All ingredients except sulfide are dissolved in deionized water and themedium is flushed with nitrogen gas (purity 99.999%) for 20 min at roomtemperature. After addition of sulfide, the pH-value is adjusted to 7.0at room temperature with 1 M HCl. The medium is then dispensed intoHungate tubes or serum flasks under nitrogen atmosphere and the vesselsare tightly sealed. After autoclaving at 121° C. for 20 min pH-valueshould be in between 6.8 and 7.0.

Carbon sources as specified for individual experiments are added priorto autoclaving. All applied substrate concentrations are indicated asglucose equivalents on the basis of available mol C (carbon).

Subsequent to autoclaving, cultures are inoculated by injection of aseed culture through the seal septum and inoculated in an incubator at72° C. for the time indicated.

Example 2 HPLC

Sugars and fermentation products were quantified by HPLC-RI using a ViaHitachi LaChrom Elite (Hitachi corp.) fitted with an Rezex ROA OrganicAcid H+ (Phenomenex). The analytes were separated isocratically with 2.5mM H₂S0₄ and at 65° C.

Example 3 Phylogenetic Analysis of 16S rDNA Genes

Genomic DNA was isolated from cultures grown as described above and16SrDNA amplified by PCR using 27F (AGAGTTTGATCMTGGCTCAG; SEQ ID No. 8)as forward and 1492R (GGTTACCTTGTTACGACTT; SEQ ID No. 9) as reverseprimer. The resulting products were sequenced and the sequences analyzedusing the Sequencher 4.10.1 software (Gene Codes Corporation). The NCBIdatabase was used for BLAST procedures.

Sequencing of 16S rDNA from all strains listed in table 1 revealed allthese had (at least) one copy of a 16S rDNA operon which was mostclosely related to Caldicellulosiruptor saccharolyticus (Strain Tp8T=DSM8903) in the available public databases. Alignment was carried outusing ClustalW (Chenna et al. 2003) and the phylogenetic tree wasconstructed using software MEGA4 (Kumar et al. 2001). The tree for allstrains listed in table 1 is displayed in FIG. 1.

The 16S rDNA sequences of all strains listed in table 1 have 99% percentidentity to the respective sequence of e.g. Caldicellulosiruptorsaccharolyticus (Strain Tp8T=DSM8903).

Example 4 Batch Experiments

Batch experiments with all strains were executed by cultivation on themedium described above with the carbon source substrates listed in FIGS.10 and 11. Sealed Hungate tubes or serum flaks were used for cultivationin a standard incubator at a temperature of 72° C.

The results clearly show that all strains are capable to producecarboxylic acids such as lactic acid and acetic and on soluble sugars,on soluble and insoluble sugar polymers as well as on the pretreatedlignocellulose in the absence of free sugars.

Physiological comparison with the strain DSM8903 identified as the mostclosely related to the 16S rDNA comparison indicates a significantlyhigher lactate formation in combination with a partially decreasedproduction of acetate on polymeric substrates.

Example 5 Fermentation

Batch experiments with all strains, e.g. DIB004C, were performed bycultivation on the medium described above with addition of 20 g/Lmiscanthus grass pretreated with a suitable method selected from thosedescribed above comprising heating in the presence of dilute acidfollowed by sudden release of pressure.

Temperature is controlled to 72° C. and the pH-value is controlled to6.75±0.1 throughout the fermentation. The fermenter is purged withnitrogen to remove excess oxygen before sodium sulphide is added asdescribed above.

The fermentation is started by addition of a seed culture prepared asdescribed in example 1.

The results of the HPLC analysis as described in example 2 show theproduction of lactate and acetate.

The results of the product formation during a fermentation ofCaldicellulosiruptor sp. DIB004C on pretreated miscanthus grass is shownin FIG. 9.

LIST OF ADDITIONAL REFERENCES

Rainey F A, Donnison A M, Janssen P H, Saul D, Rodrigo A, Bergquist P L,Daniel R M, Stackebrandt E, Morgan H W. (1994) Description ofCaldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligatelyanaerobic, extremely thermophilic, cellulolytic bacterium. FEMSMicrobiol Lett. 120:263-266.

Sissons C H, Sharrock K R, Daniel R M, Morgan H W. (1987) Isolation ofcellulolytic anaerobic extreme thermophiles from New Zealand thermalsites. Appl Environ Microbiol. 53:832-838.

Donnison A M, Brockelsby C M, Morgan H W, Daniel R M. (1989) Thedegradation of lignocellulosics by extremely thermophilicmicroorganisms. Biotechnol Bioeng. 33:1495-1499.

Hungate R E. (1969) A roll tube method for cultivation of strictanaerobes. In: Methods in Microbiology Eds. Norris J R and Ribbons D W.pp 118-132. New York: Academic Press.

Chenna R, Sugawara H, Koike T, Lopez R, Gibson T J, Higgins D G,Thompson J D. (2003) Multiple sequence alignment with the Clustal seriesof programs. Nucleic Acids Res. 13:3497-3500.

Kumar S, Tamura K, Jakobsen I B, Nei M. (2001) MEGA2: molecularevolutionary genetics analysis software. Bioinformatics. 17:1244-1245.

1. A method for converting lignocellulosic biomass material to acarboxylic acid comprising the step of contacting the lignocellulosicbiomass material with a microbial culture for a period of time at aninitial temperature and an initial pH, thereby producing an amount of aa carboxylic acid; wherein the microbial culture comprises an extremelythermophilic bacteria strain of the genus Caldicellulosiruptor, whereinthe lignocellulosic biomass material is converted in a single stepprocess as part of a consolidated bioprocessing (CBP) system. 2.(canceled)
 3. The method according to claim 1, wherein the period oftime is 10 h to 300 h, preferably 50 h to 200 h, 80 h to 160 h.
 4. Themethod according to claim 1, wherein the initial temperature is in therange between 55° C. and 80° C., optionally between 72° C. and 78° C. 5.The method according to claim 1, wherein the initial pH is between 5 and9, optionally between 6 and
 8. 6. The method according to claim 1,wherein the carboxylic acid is lactic acid and/or acetic acid, salts oresters thereof.
 7. The method according to claim 1, wherein thelignocellulosic biomass material is selected from the group consistingof grass, switch grass, cord grass, rye grass, reed canary grass, mixedprairie grass, miscanthus, sugar-methoding residues, sugarcane bagasse,sugarcane straw, agricultural wastes, rice straw, rice hulls, barleystraw, corn cobs, cereal straw, wheat straw, canola straw, oat straw,oat hulls, corn fiber, stover, soybean stover, corn stover, forestrywastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, andsoftwood, pressmud from sugar beet, cotton stalk, banana leaves, andlignocellulosic biomass material obtained through processing of foodplants.
 8. (canceled)
 9. The method according to claim 1, wherein saidlignocellulosic biomass material is selected from the group consistingof corn stover, sugarcane bagasse, cotton stalks, switchgrass, andpoplar wood. 10-13. (canceled)
 14. The method according to claim 1,wherein said lignocellulosic biomass material is a grass or perennialgrass, optionally miscanthus.
 15. (canceled)
 16. (canceled)
 17. Themethod according to claim 1, wherein said lignocellulosic biomassmaterial is subjected to mechanical, thermochemical, and/or biochemicalpretreatment.
 18. The method according to claim 17, wherein pretreatingthe lignocellulosic biomass material comprises exposing thelignocellulosic biomass to steam treatment.
 19. The method according toclaim 17, wherein pretreating the lignocellulosic biomass materialcomprises mechanical commination and a subsequent treatment withsulfurous acid or its anhydride under heat and pressure with a suddenrelease of pressure.
 20. The method according to claim 17, whereinpretreating the lignocellulosic biomass comprises milling thelignocellulosic biomass.
 21. (canceled)
 22. The method according toclaim 17, wherein the lignocellulosic biomass material is pretreated inaddition with enzymes, preferably cellulose and hemicellulose degradingenzymes.
 23. The method according to claim 1, wherein the strain isselected from the group consisting of Caldicellulosiruptor sp. DIB041C,deposited as DSM 25771, Caldicellulosiruptor sp. DIB087C, deposited asDSM 25772, Caldicellulosiruptor sp. DIB103C, deposited as DSM 25773,Caldicellulosiruptor sp. DIB104C, deposited as DSM 25774,Caldicellulosiruptor sp. DIB107C, deposited as DSM 25775,Caldicellulosiruptor sp. DIB 101 C, deposited as DSM 25178 andCaldicellulosiruptor sp. DIB004C, deposited as DSM 25177, microorganismderived therefrom, progenies or mutants thereof.
 24. (canceled) 25.(canceled)